1. Field of the Invention
The present invention relates to biocontrol of plant root diseases. In particular, the invention relates to strains of fluorescent Pseudomonas species which have a biosynthetic locus which encodes for the production of the antibiotic phenazine-1-carboxylic acid stably introduced into the genome, and have biocontrol activity for control of plant root diseases, in particular, diseases caused by the soil-borne pathogen, Rhizoctonia. The invention further relates to methods of making the transgenic strains, and application thereof to control plant root diseases.
2. Description of the Art
Root diseases caused by Rhizoctonia, Pythium, and Gaeumannomyces graminis, cause a significant adverse impact on the production of important crops worldwide. The root disease take-all, caused by Gaeumannomyces graminis var. tritici (Ggt), Rhizoctonia root rot, caused by Rhizoctonia solani and R. oryzae, and Pythium root rot caused by any of several Pythium species, notably, Pythium ultimum and P. irregulare, are important root diseases of small grain crops, e.g., wheat, barley, triticale, and rye, worldwide.
Rhizoctonia, a member of the basidiomycotina class of fungi, causes root and stem rot on most food, fiber, and ornamental plants throughout the world, including small grain crops, turf grass, asparagus, canola, corn, sugarbeet, tomatoes, potatoes, peas, rice, beans, soybeans, strawberries, zucchini, and cotton. Root rot on small grain crops caused by Rhizoctonia occurs throughout the United States Pacific Northwest, in Australia, and South Africa, and potentially throughout the temperate regions of the world wherever small grains are grown, especially if grown with reduced or notillage (direct drilling). Rhizoctonia root rot caused by R. solani AG8 begins as brown cankerous lesions on the seminal and crown roots that eventually girdles and then severs the roots. Plants with roots pruned off by this disease remain stunted and eventually die without making heads. The disease tends to affect plants in patches and has given rise to other names, such as bare patch disease, purple patch, crater disease, and barley stunt disorder. Of all small grain crops, barley is especially susceptible to R. solani AG8. Rhizoctonia oryzae infects the embryos of germinating seeds, preventing germination or limiting the formation of seminal roots to only one or two when healthy seedlings produce five or six seminal roots. These two Rhizoctonia species, together with Rhizoctonia cerealis and possibly other Rhizoctonia species occur as different mixtures, depending on the soil, cropping systems, weed management practices, and possibly other factors not yet identified.
The soil-borne pathogen complex of Pythium spp. comprises a group of fungi that are among the most successful of all microbial colonists in agricultural soils. It is estimated that nearly all cultivated soil in the world contains spores of at least one, two, three, and even as high as ten Pythium species. Pythium, a member of the oomycetes class of fungi, like Rhizoctonia, affects virtually all food, fiber, and ornamental plants throughout the world. Examples of these plants are given above. Pythium damage to small grains begins as embryo infections and associated poor emergence or stand establishment and continues as destruction of the fine lateral rootlets and root hairs. Plants with Pythium root rot have the appearance of plants without enough fertilizer, because the disease limits the absorptive capacity of the root system through destruction of fine rootlets and root hairs. There are several species of Pythium with ability to attack cereals, either embryos of germinating seeds, root tips and fine rootlets, or all of these delicate and usually juvenile or meristematic tissues.
Widespread diseases of small grain crops and turf grass are caused by the soil-bome fungus Gaeumannomyces graminis (Gg), a member of the ascomycotina class of fungi, and result in significant economic losses due to reductions in crop yield. Take-all, a disease caused by Gaeumannomyces graminis var. tritici (Ggt) occurs in all wheat-growing regions of the world and is probably the most important root disease of wheat and related small grains worldwide. Symptoms of wheat take-all include dark longitudinal lesions on roots; in severe cases, the entire root may become blackened with disease with the fungus migrating to the crown of the wheat plant (where the crown roots originate) and the tillers (stems). Severely infected wheat plants are identified in the field by their white heads which result when infection of the crown by the fungus cuts off water transport to upper plant parts causing the plant to die prematurely. Yield losses can be considerable up to 50% of the potential wheat yield. There are no resistant wheat cultivars and registered fungicides perform inconsistently. Further, growers are being increasingly challenged to grow wheat with minimum or no tillage to reduce soil erosion. These practices increase the severity of take-all and other root diseases. Although wheat is particularly susceptible to the take-all fungus, many other Gramineae such as barley, rye, and triticale can also be infected.
Traditionally, take-all has been controlled by a combination of crop rotation and tillage, practices which reduce the inoculum potential of the pathogen. However, because long rotations are often not economically feasible and tillage contributes to soil erosion, the trend in cereal production is toward less tillage and two or three wheat crops before a break. Both of these practices exacerbate take-all. There is no known source of genetic resistance in wheat against take-all, and methods of chemical control are limited. The need for agriculture to become more sustainable and less dependent on chemical pesticides has necessitated the development of alternative approaches to control take-all and other soil-borne diseases.
Other Gg fungi, for example, Gaeumannomyces graminis var. avenae (Gga) infects oats and grasses and have been identified as causing take-all patch in turf grasses such as bent grass. Gaeumannomyces graminis var. graminis (Ggg) infects some grases and has been suggested as causing crown sheath rot in rice.
The pathogens responsible for takeall and Rhizoctonia root rot survive as hyphae or mycelium in the tissues of host plants colonized through their parasitic activities. Pythium species survive in soil as thick-walled oospores or sporangia produced from nutrients robbed from the plant through parasitism. Usually, all three diseases develop simultaneously on the same plants, although one root disease may dominate.
Although Pythium species are ubiquitous in agricultural soils cropped to small grains, damage to small grains caused by Pythium species, e.g., reduction in seedling emergence and plant vigor, is greatest in soils kept wet, especially if the soils are also naturally high in clay content and with pH values below 6.0. Allowing volunteer cereals (plants that develop from seed spilled or dropped by the harvester on the soil surface) to grow in the field after harvest of one crop until only 1 or 2 days before planting the next crop, then spraying with an herbicide such as glyphosate (Round-up.RTM., Monsanto), controls the weeds but greatly favors Pythium root rot and Rhizoctonia root rot. Planting wheat directly into the standing stubble of a previous wheat crop with soil kept moist by sprinkler irrigation or leaving the soil covered with straw favors all three root diseases.
Wheat and other cereals with root disease yield poorly and return less on investments to the grower. Plants with these root diseases also compete poorly with weeds, thereby making it necessary to spend more on herbicides to control weeds. Small grains with root diseases also leave fertilizer unused in the soil, including nitrates, which then may move by leaching below the rooting zone and eventually into ground water. Growers throughout the world continue to use some form of tillage for production of small grains, largely because tillage helps control these root diseases. Tillage causes soils to be more vulnerable to soil erosion. It also requires more energy, and leads to greater evaporation of water needed for yield. Some farmers, attempting to use no-till, burn the stubble in their fields in the belief that this will provide some relief from root diseases. Stubble burning is both environmentally detrimental and socially unacceptable, especially to people in urban areas and cities that object to having to breathe the smoke produced by stubble burning.
Many diseases of wheat, barley, and other crops are controlled by breeding varieties of the crops with resistance to the pathogens. However, this approach has worked mainly for leaf diseases but not for root diseases of wheat, barley, triticale or rye. The only known source of resistance to take-all and Rhizoctonia root rot is in a very distant diploid relative, Daysapyrun villosum, but thus far no use has been made of this source of resistance because of the difficulty of transferring genes across such a taxonomically wide distance. No commercial wheat, barley, rye or triticale exists at the present time in the world with resistance to take-all, Rhizoctonia root rot, or Pythium root rot.
Methods available for biological control of fungal pathogens on plants have included bacterial strains of the species Pseudmonas having pathogen-specific activity. U.S. Pat. No. 4,456,684 describes Pseudomonas strains which suppress disease caused by take-all and other Gg fungi. Studies of the microbial antagonism involved in take-all decline, a natural biological control of take-all, defined as the spontaneous reduction in disease and the increase in yield with extended monoculture of Ggt-susceptible small grain crops such as wheat and barley, have focused on attempts to identify specific Ggt-antagonistic microorganisms and to transfer these organisms to soil to reproduce suppression. Many of the most effective strains produced the antibiotic 2,4-diacetylphloroglucinol (Phl) (C. Keel et al., Applied and Environmental Microbiology 62:552-563 (1996)). J. M. Raaijmakers et al. (Applied and Environmental Microbiology 63:881-887 (1997)) report that Phl-producing fluorescent Pseudomonus spp. were present on roots of wheat grown in three TAD soils from Washington State (USA). Although use of microbial biocontrol agents holds great promise as a practical means to control soilborne pathogens, all published or patented biocontrol agents for take-all have the disadvantages of performing inconsistently, being soil-specific, and being unable to duplicate the level of control consistently observed in a TAD soil.
U.S. Pat. No. 4,647,533 reports Pseudomonas strains which suppress diseases caused by Pythium. Strains of Pseudomonas bacteria inhibitory to either Rhizoctonia solani or Pythium ultimum on cotton have been reported. (See U.S. Pat. No. 5,348,742 to Howell et al.) Bacillus sp. L324-92 has been reported to simultaneously control Gaeumannomyces graminis, Rhizoctonia and Pythium species (Kim et al. Phytopathology 87:551-558 (1997)). However, no single Pseudomonas strain has been reported that is effective in controlling all three of these pathogens.